There is an increasing need in biological research, and more particularly diagnostic medicine, for identifications and characterizations of nucleic acids. By "nucleic acids" there are to be understood in the present case the deoxyribose nucleic acids (DNA) and the ribose nucleic acids (RNA) in either naturally occurring form or as they can be produced by modern methods of chemical and biological synthesis of substantially any sequence and length.
Conventional methods used in molecular biology to prepare nucleic acids from blood are complex and include steps such as centrifuging, phenol/chloroform extraction of the samples or precipitations of the nucleic acids with organic solvents, which are useless for rapid and possibly automatable enzymatic amplification of nucleic acids without substantial preparation. A recent compilation of such methods is found in "An efficient and simple method of DNA extraction from whole blood and cell-lines to identify infectious agents" by V. N. Loparev et al., J. Vir. Methods 34:105-112 (1991) and in "Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material" by P. S. Walsh et al., BioTechniques 10(4):506-513 (1991).
It has been reported in the literature that whole blood inhibits the polymerase chain reaction (PCR) when present even in very small quantities -- i.e., 1 volume % in the reaction mixture. The reason for this inhibition is believed to be due to heme derivatives consisting of porphyrin rings that are present in blood (R. Higuchi, PCR Technology, Chapter "Simple and rapid preparation of samples for PCR", pages 31-38, H. Ehrlich Ed., Stockton Press, 1989).
According to Higuchi, supra, a method of preparing target DNA molecules in blood samples for PCR is to isolate the mononuclear blood cells (MC) by way of ficoll gradients or to isolate the leucocytes by centrifuging after lysis of the erythrocytes and to incubate the MCs with proteinase K. After digestion, the proteinase K is inactivated at 95.degree. C. and an aliquot of the sample is used in the PCR process.
Mercier et al. describe PCR amplification of various fragments of chromosomal DNA from fresh blood or frozen blood in a concentration of 1 to 2 volume % in a PCR reaction mixture (Nucleic Acids Research 18:5908 (1990)) in which the amplification solution containing the blood sample (without Taq polymerase) was repeatedly brought for 3 minutes at a time to temperatures of 95.degree. C. and 55.degree. C., a step which facilitated subsequent amplification by PCR.
Panaccio et al. (Nucleic Acids Research 19:1151 (1991)) describe the amplification of DNA from whole blood using thermostable DNA polymerase from Thermus thermophilus. They show that DNA from 4 .mu.l of blood in 100 .mu.l of reaction mixture (4 volume %) is amplifiable using Thermus thermophilus DNA polymerase. On the other hand, as little as 1 volume % of blood completely inhibits amplification by means of the DNA polymerase from Thermus aquaticus (Taq).
Beutler et al. (BioTechniques 9:166 (1990)) describe in detail the effect of anticoagulants on the amplification by PCR of DNA targets from blood samples. It was impossible to amplify these targets even when the DNA was concentrated from heparinized blood by means of a nucleic acid extract. Further processes for purifying DNA that facilitate use of PCR with the DNA thus isolated are also described, including the treatment of DNA with heparinase II. There were no problems with amplifying DNA isolated from EDTA-treated blood.
Israeli et al. (Nucleic Acids Research 19:6051 (1991)) describe the amplification by PCR of RNA which was isolated from frozen heparin-treated whole blood by extraction after conversion of the RNA into eDNA. Israeli demonstrate that the difficulties in conducting PCR were due to the heparin. Only when the isolated RNA was treated with heparinase before transcribing into eDNA, was the PCR successful.
Franchis et al. (Nucleic Acids Research 16:10355 (1988)) also describe the inhibition of the PCR process using Taq polymerase when amplifying samples of genomic DNA isolated from human blood. The inhibitor, which was not specifically identified in the article, could be removed by boiling and filtering the DNA.
Ravaggi et al. (PCR Methods and Applications 4:291-292 (1992)) describe the amplification of HCV RNA from human serum by means of PCR. The RNA was transcribed directly from the serum into eDNA with reverse transcriptase without previous purification. An aliquot containing approximately 3 volume % of DNA then was introduced into a PCR mix.
One of the goals of this invention is to overcome the difficulties noted in the prior art described above in order to use increased quantities of blood directly in an enzymatic amplification process for nucleic acids, such as, for example, PCR, and more particularly, when the blood is treated with anticoagulants.